Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca²⁺ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca²⁺ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca²⁺ entry and subsequent endothelium-dependent dilation in human arterioles.

受损的内皮依赖性血管舒张被认为是冠状动脉微血管功能障碍 (CMD) 的关键组成部分。更好地了解参与人体小动脉血管舒张的内皮通路可能会为 CMD 的机制提供新的见解。本研究的目的是研究 TRPV4、NOX4 的作用及其在人体小动脉中的相互作用，并研究其潜在机制。从 71 名没有冠状动脉疾病的患者的脂肪和心脏组织中新鲜分离出小动脉，并通过视频显微镜研究了血管反应性。在人脂肪小动脉 (HAA) 中，ACh 诱导的扩张被 TRPV4 抑制剂 HC067047 和 NOX 1/4 抑制剂 GKT137831 显着降低，但 GKT137831 不会进一步影响存在 TRPV4 抑制剂的扩张。GKT137831 还抑制 TRPV4 激动剂 GSK1016790A 诱导的 HAA 和人冠状动脉 (HCA) 扩张。在 HAA 和 HCA 的内皮细胞中检测到 NOX4 转录物和蛋白质。使用 fura-2 成像，GKT137831 显着降低 GSK1016790A 诱导的 Ca²⁺流入内皮细胞和 TRPV4-WT 过表达人冠状动脉内皮细胞 (HCAEC) 的原代培养物中。然而，GKT137831 不影响非磷酸化 TRPV4-S823A/S824A 过表达 HCAEC 中TRPV4 介导的 Ca ^2+流入。此外，用 GKT137831 处理 HCAEC 可降低 TRPV4 中 Ser824 的磷酸化水平。最后，邻近连接分析 (PLA) 揭示了 NOX4 和 TRPV4 蛋白的共定位。总之，TRPV4 和 NOX4 都有助于 ACh 诱导的无冠状动脉疾病患者的人小动脉扩张。NOX4 增加内皮细胞中的 TRPV4 磷酸化，进而增强 TRPV4 介导的 Ca ²⁺进入和随后的人小动脉内皮依赖性扩张。